66 5 ART for Anti-inflammation
intra-articular CFA injection and LIAA mice established by intra-articular LPS
injection also show the morphological and histological lesions that resemble CICA
mice established by intradermal CII-CFA injection. In general, synovitis occurs
in CICA mice within weeks after twice injections with CII-CFA, but it occurs in
CIAA or LIAA mice only within three days upon only one injection with CFA or
LPS. These acute synovitis/arthritis models should represent the most rapid mod-
eling procedure of RA in mice so far.
To elucidate the relevance of arthritic modeling with immune activation, we
investigated the cytokine microarray profiles in CIAA mice upon pre- and postim-
munization. Consequently, 31 cytokines are upregulated in CIAA mice, including
the proinflammatory cytokines IFNγ (1.5-folds), TNFα (2.24-folds), and IL-1β
(1.46-folds). In similar, LPS injection also increases the serum levels of TNFα to
1.691 ± 0.07 pg/ml in LIAA mice, which is very significantly different from con-
trol mice (1.128 ± 0.07 pg/ml). These results clearly demonstrated that intra-artic-
ular injection with CFA or LPS can activate the critical proinflammatory cytokines
and initiate the global inflammatory responses.
5.3.2.2 NO Production, Hypoxia, and Angiogenesis in CIAA
and LIAA Mice
After intra-articular injection of mice with CFA, we observed the generation of
NO after only 4 h, and determined the accumulation of NO after one day. Potent
NO burst occurs on the 2nd day, and the NO peak (above 10 μM) slightly declines
on the 3rd day. Accordingly, CIAA mice exhibit a dramatic decrease of SpO 2 ,
immediately after CFA injection. While control mice have a maximal SpO 2 above
80 %, CIAA mice have a minimal SpO 2 below 60 %. In LIAA mice, NO also
reversely correlates with SpO 2 , in which the NO level elevates from 2 μM (con-
trol mice) to 8 μM, and SpO 2 declines from 98.33 ± 0.58 % (control mice) to
67.00 ± 1.73 %. The elevation of NO is followed by the decline of SpO 2 , imply-
ing that a high NO level might lead to a low SpO 2 upon modeling.
To reveal whether CFA would affect the expression of HIF-1α and VEGF, we
performed the immunohistochemical analysis on synovial sections prepared from
CIAA mice. Consequently, both of which are significantly upregulated after CFA
injection, although HIF-1α shows a higher expression level than that of VEGF.
Furthermore, CFA was observed to promote the significant synovial angiogen-
esis. In LIAA mice, HIF-1α and VEGF are markedly upregulated, in which the
immunohistochemical staining strength of HIF-1α is 99.96 ± 1.17 (11.81 ± 1.95
in control mice), and that of VEGF is 110.68 ± 4.55 (9.04 ± 0.08 in control mice)
(Fig. 5.2). These results indicated that immunization-triggered NO and NO-driven
hypoxia can upregulate HIF-1α and VEGF. Accordingly, we observed the eleva-
tion of serum LA levels on the 1st day of CFA injection, but they gradually decline
latter, suggesting that hypoxia-mediated glycolysis is inhibited and angiogenesis is
activated following the induction of HIF-1α and VEGF.